Signal normalization of nucleic acid amplification reaction products
OwnerStefan Otto Corneliusinventor
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Abstract
The invention relates to a multiplex nucleic acid amplification reaction comprising at least two separate amplicons, with normalized signal strength, wherein for at least one amplicon either the forward primer oligonucleotides, or the reverse primers oligonucleotides, or the optional detection probe oligonucleotides, are only partially labeled, and the subset amount of labeled primer/probe oligonucleotides to subset amount of unlabeled primer/probe oligonucleotides is adjusted so that the signal strength in both amplicons is equal or nearly equal. Kits and compositions are also claimed.
Claims12
Multiplex nucleic acid amplification reaction comprising at least two separate amplicons, with normalized signal strength, wherein
- for at least one amplicon either the forward primer oligonucleotides, or
- the reverse primers oligonucleotides, or the optional detection probe oligonucleotides, are only partially labeled, and
- the subset amount of labeled primer/probe oligonucleotides to subset amount of unlabeled primer/probe oligonucleotides is adjusted so that the signal strength the at least two amplicons is equal or nearly equal,
- wherein the amplification reaction reaches the plateau phase.
Reaction according to any of the preceding claims, wherein the amplicons are located on one or more template nucleic acids and the amount of the one or more template nucleic acids in said nucleic acid amplification reaction mixture is between 1 fg to 5 μg, more preferably between 100 fg to 1 μg, even more preferably between 500 fg to 500 ng, even more preferably between 750 fg to 250 ng, even more preferably between 850 fg to 250 ng, even more preferably between 900 fg to 150 ng, even more preferably between 1 μg to 150 ng and most preferably between 2 μg to 110 ng.
Reaction according to any of the preceding claims, wherein detecting the labels of the first subset of oligonucleotides is performed in capillary electrophoresis, gel electrophoresis, pyrosequencing, sanger sequencing, next generation sequencing, digital PCR, real-time PCR, quantitative PCR, isothermal PCR, or in microarray analysis.
Reaction according to any of the preceding claims, wherein the reaction is an endpoint PCR, a digital PCR, a real-time PCR, a quantitative PCR, an isothermal PCR, a loop-mediated isothermal amplification, a recombinase polymerase amplification, a nicking enzyme amplification reaction, a nicking endonuclease signal amplification, a rolling circle amplification, a helicase-dependent amplification, a hybridization chain reaction, a multidisplacement amplification, an isothermal assembly reaction or any combination thereof.
Reaction according to any of the preceding claims for use in STR analysis, SNP analysis, genotyping, molecular diagnostics, genetic research or population genetics.
Dependencies are inferred from the claim text.